CN109022588A - A kind of grouper stocked population identifies specific primer and the application of microsatellite marker - Google Patents
A kind of grouper stocked population identifies specific primer and the application of microsatellite marker Download PDFInfo
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Abstract
The invention discloses the specific primers that a kind of grouper stocked population identifies microsatellite marker, the specific primer is 12 pairs, the specific primer of specific primer and 5 couple of four base repetitive sequence GRQU-1~GRQU-5 including 7 couple of three base repetitive sequence GRTR-1~GRTR-7, wherein the base sequence of the specific primer of three base repetitive sequence GRTR-1~GRTR-7 is as shown in No:1~14 SEQ ID, and the base sequence of the specific primer of four base repetitive sequence GRQU-1~GRQU-5 is as shown in No:15~24 SEQ ID.A kind of grouper stocked population mirror method for distinguishing and above-mentioned specific primer are also disclosed in the paternity test of grouper stocked population, group's identification and genetic breeding and releases the application in recruitment evaluation.
Description
Technical field
The invention belongs to DNA molecular marker technical fields, and in particular to a kind of grouper stocked population identification microsatellite mark
The specific primer of note and application.
Background technique
Grouper is under the jurisdiction of Lu Xingmu Sushi section, is rare seawater fish, is also important cultured fishes, and some types are for example oblique
Band grouper, epinephelus lanceolatus fish, epinephelus fuscoguttatus etc. have cultivated extensively in China and south east asia.In recent years, due to mistake
Factors, the wild stocks stock number of ablen such as degree fishing and environmental disruption sharply decline, and fish-farming operation department has to using increasing
It grows the mode released and protects its natural resources.
Although enhancement releasing effectively can restore and increase the population quantity of natural water area, there is also biggish ecological wind
Danger, may genetic diversity to waters wild stocks, ecological grade of fit have an impact.Therefore, to release effect into
Row needs long-term tracking and monitoring, and key among these is to identify stocked population, and then ability from recapture group
Analyze stocked population survival in the natural environment and diffusion-condition, and the influence to local wild stocks.
The common label for identifying stocked population includes being listed, cutting fin, branding and fluorchrome etc., and there are label retention
It is low, influence label fish behaviors and growth and the disadvantages of.
DNA molecular marker is a kind of novel labelling technique, and detection is hereditary difference on DNA level, is not only overcome
Above-mentioned label disadvantage, and polymorphism is high, and group identifies more efficient.
Only have a few species such as lefteye flounder, egg-shaped pompano and sparid class in seawater fish at present and apply molecular labeling and is increasing
It grows and releases, there is not yet the correlative study of ablen is reported.It would therefore be highly desirable to which constructing a set of can quick and precisely identify grouper and release
The molecular labeling specific primer of group provides pass for the assessment of the enhancement releasing hereditary effect of ablen and other seawater fish
The technical support of key.
Summary of the invention
The present invention provides one for not having also grouper stocked population to identify the specific primer of microsatellite marker at present
Kind grouper stocked population identifies the specific primer of microsatellite marker.
The object of the invention is also to provide a kind of discrimination methods of grouper stocked population.
Third object of the present invention is that the specificity for providing above-mentioned grouper stocked population identification microsatellite marker is drawn
Object is in the paternity test of grouper stocked population, group's identification and genetic breeding and releases the application in recruitment evaluation.
Above-mentioned first purpose of the invention is realized by the following technical means: a kind of grouper stocked population mirror
The specific primer of other microsatellite marker, the specific primer are 12 pairs, including 7 couple of three base repetitive sequence GRTR-1~
The specific primer of the specific primer of GRTR-7 and 5 couple of four base repetitive sequence GRQU-1~GRQU-5, wherein three base weights
For the base sequence of the specific primer of complex sequences GRTR-1~GRTR-7 as shown in No:1~14 SEQ ID, four bases repeat sequence
The base sequence of the specific primer of GRQU-1~GRQU-5 is arranged as shown in No:15~24 SEQ ID.
The present invention searches " polybase base weight multiple " microsatellite markers from Epinephelus coioides genome, by specificity and
Polymorphism etc. is repeatedly screened, and 12 " polybase base weight multiple " (including 7 three bases and 5 four base repetitive sequences) micro- is obtained
Satellite markers, the expectation heterozygosity of label are 0.396~0.803, and polymorphism information content is 0.346~0.772, show label
Polymorphism is higher.
Above-mentioned 12 pairs of specific primers be include 7 couple of three base repetitive sequence GRTR-1~GRTR-7 specific primer and
The specific primer of 5 couple of four base repetitive sequence GRQU-1~GRQU-5.
Above-mentioned second purpose of the invention is realized by the following technical means: a kind of grouper stocked population
Discrimination method, comprising the following steps:
(1) grouper " polybase base weight is multiple " microsatellite marker is screened, above-mentioned specificity is designed according to microsatellite marker and is drawn
Object；
(2) using the specific primer in step (1), grouper " polybase base weight is multiple " microsatellite marker multiplex PCR is established
System；
(3) by grouper stocked population and its parental population, using the multiplex PCR system established in step (2), in whole
It is expanded in individual, then carries out paternity test；
(4) it is different next that wild population composition the identification verifying of grouper stocked population: is added in grouper stocked population
The population mixture in source discriminates stocked population based on paternity test technology using the multiplex PCR system established in step (2)
Not.
In grouper stocked population mirror method for distinguishing:
In step (1) of the present invention: it is " two base weights mostly that stocked population, which identifies microsatellite molecular marker used, at present
It is multiple " sequence, compared with the duplicate microsatellite marker of polybase base, there are resolution ratio is low and shadow for " repetition of two bases " microsatellite marker
With more problems, the accuracy of Genotyping is influenced.
The present invention searches " polybase base weight multiple " microsatellite markers from Epinephelus coioides genome, by specificity and
Polymorphism etc. is repeatedly screened, and 12 " polybase base weight multiple " (including 7 three bases and 5 four base repetitive sequences) micro- is obtained
Satellite molecule label, the expectation heterozygosity of label are 0.396~0.803, and polymorphism information content is 0.346~0.772, show to mark
The polymorphism of note is higher.
According to three base sequences filtered out, four base repetitive sequence microsatellite locus synthesis fluorescent primer to get special
Property primer, be 12 pairs, base sequence is as shown in No:1~24 SEQ ID.
Grouper " polybase base weight is multiple " microsatellite marker multiplex PCR system is established in step (2) of the present invention, respectively by three
Base sequence, four base sequences are expanded in Epinephelus coioides stocked population individual, since to will appear primer mutually dry for multiplex PCR
Disturb, non-specific amplification and small fragment expanding effect are uneven better than amplification caused by large fragment etc., the present invention passes through primer
The condition optimizings such as concentration and annealing temperature, be finally obtained amplification preferably, the grouper of stable and uniform " polybase base weight is multiple "
Microsatellite marker multiplex PCR system.
Multiplex PCR can be in a PCR reaction, while amplifying multiple microsatellite locus, time saving and energy saving efficient.
Preferably, microsatellite marker multiplex PCR system described in step (2) includes two multiplex PCR systems, wherein more
The primer that weight PCR system 1 uses is adopted for the specific primer of three base repetitive sequence GRTR-1~GRTR-7, multiplex PCR system 2
Primer is the specific primer of four base repetitive sequence GRQU-1~GRQU-5.
Preferably, the total volume of multiplex PCR system 1 is 20 μ L, including 2x Multiplex PCR MasterMix10 μ L,
GRTR-1 specific primer (positive and negative primer) totally 0.5 μ L (0.25 μM), GRTR-2 specific primer (positive and negative primer) totally 0.6 μ L
(0.3 μM), GRTR-3 specific primer (positive and negative primer) totally 0.45 μ L (0.225 μM), GRTR-4 specific primer is (positive and negative to draw
Object) totally 0.3 μ L (0.15 μM), GRTR-5 specific primer (positive and negative primer) totally 0.55 μ L (0.275 μM), GRTR-6 specificity
Primer (positive and negative primer) totally 0.3 μ L (0.15 μM), GRTR-7 specific primer (positive and negative primer) totally 0.2 μ L (0.1 μM), DNA
Template (100ng/ μ L) 1 μ L, ddH2O 6.1μL。
Preferably, the total volume of multiplex PCR system 2 is 20 μ L, including 2x Multiplex PCR MasterMix10 μ L,
GRQU-1 specific primer (positive and negative primer) totally 0.3 μ L (0.15 μM), GRQU-2 specific primer (positive and negative primer) totally 0.35 μ
L (0.175 μM), GRQU-3 specific primer (positive and negative primer) totally 0.25 μ L (0.125 μM), GRQU-4 specific primer is (just,
Anti-primer) totally 0.25 μ L (0.125 μM), GRQU-5 specific primer (positive and negative primer) totally 0.35 μ L (0.175 μM), DNA profiling
7.5 μ L of (100ng/ μ L) 1 μ L, ddH2O.
Preferably, the reaction condition of microsatellite marker multiplex PCR system described in step (2) are as follows: 95 DEG C of initial denaturations
10min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 1min, totally 35 recycle, last 72 DEG C of extensions 5min.
Step (3) of the present invention shows that " polybase base weight the is multiple " microsatellite molecular marker of acquisition and multiplex PCR system can be accurate
The paternity test of grouper is carried out, therefore can be applied to the genetic breeding research of grouper.
Step (4) of the present invention shows that " polybase base weight the is multiple " microsatellite molecular marker of acquisition and multiplex PCR system can be accurate
The group for carrying out grouper identifies, therefore can be applied to grouper releases recruitment evaluation.
Above-mentioned third purpose of the invention is realized by the following technical means: above-mentioned grouper stocked population
Identify paternity test of the specific primer in grouper stocked population, group's identification and the genetic breeding of microsatellite marker and puts
Flow the application in recruitment evaluation.
The invention has the following advantages that the present invention all uses " polybase base weight is multiple " microsatellite molecular marker and its specificity
Primer, high resolution and shadow band are few, and Genotyping accuracy is high；Multiplex PCR system amplification after optimization is preferable, stable
Uniformly；The affiliation of accurate retrospect stocked population, accuracy rate is up to 100%；Accurate to identify stocked population, accuracy rate reaches
98.68%；This method is efficient, easy, accurate.
Detailed description of the invention
Fig. 1 is the gene that 7 three bases repeat microsatellite marker GRTR-1~GRTR-7 multiplex PCR system in embodiment 2
Parting figure；
Fig. 2 is the gene that 5 four bases repeat microsatellite marker GRQU-1~GRQU-5 multiplex PCR system in embodiment 2
Parting figure；
Fig. 3 is that the accumulation that 12 polybase base weights answer microsatellite molecular marker in embodiment 2 excludes success rate.
Specific embodiment
Embodiment 1
1, grouper polybase base weight answers microsatellite locus screening and design of primers:
It is sequenced by Illumina and obtains Epinephelus coioides genome, then carry out statistical with 3.4 software of SciRoKo
The data such as microsatellite locus quantity, type and the length in genome are analysed, setting parameter is MISA mode: 20-10-7-5-4-4.
It selects 3-4 base to repeat microsatellite sequence, uses 5.0 software Design primers of Primer Premier.
Parameter selection are as follows: length 17-27bp, optimum length 20bp；57-63 DEG C of annealing temperature；G/C content 40%-60%；
Amplified production length 100-450bp；Other parameters are set according to software default.
The specific primer that the grouper stocked population of acquisition identifies microsatellite marker is 12 pairs, base sequence such as SEQ
Shown in No:1~24 ID and shown in table 1.
2, grouper polybase base weight answers the specificity and polymorphism screening of microsatellite marker:
Specific amplification is examined first, removes non-specific amplification, the microsatellite locus that master tape is unclear, miscellaneous band is more, so
After carry out polymorphic detection, select allele and be greater than 5 microsatellite locus.
Pcr amplification reaction system are as follows: 10 μ L 2x Multiplex PCR MasterMix, positive reaction primer is (from genome
Primer of the data from 77 microsatellite locus of primary election) 17 μ L of μ L, ddH2O of (10 μm of ol/L) each 1 μ L, DNA (100ng/ μ l).
PCR program setting is as follows: 95 DEG C of initial denaturations 10min, 95 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 1min,
Totally 35 circulations, last 72 DEG C of extensions 5min.
PCR product carries out capillary electrophoresis detection genotype, calculates genetic diversity using Cervus v3.0.3 software and joins
Number.
By the repeatedly screening such as specificity and polymorphism, obtains higher 7 three bases of polymorphism and 5 four bases repeat
Sequence microsatellite marker, the number of alleles of label is 6-12, it is expected that heterozygosity is 0.396~0.803, polymorphism information content is
0.346~0.772 (table 1).
(7 three bases repeat microsatellite to the hereditary feature of 1 grouper of table 12 " polybase base weight is multiple " microsatellite molecular marker
Repeating microsatellite marker labeled as GRTR-1~GRTR-7,5 four bases is GRQU-1~GRQU-5)
3, the foundation of grouper " polybase base weight is multiple " microsatellite marker multiplex PCR system:
It is time-consuming and laborious that single microsatellite marker carries out PCR amplification detection group one by one, with multiple primers simultaneously in a PCR
Coamplification (multiplex PCR) high degree reduces cost in reaction, improves efficiency, and reduces Genotyping mistake.
Three base sequences filtered out, four base repetitive sequence microsatellite locus are synthesized into fluorescent primer (spy above
Specific primer), then select the carry out multiplexed PCR amplification with 100bp~400bp clip size difference.
The conditions such as each primer concentration in multiplex PCR system, which are adjusted, according to result obtains the multiplex PCR of uniform and stable amplification
System, optimization after annealing temperature are 60 DEG C, primer concentration (table 2, Fig. 1-2) between 0.1~0.3 μm of ol.
2 grouper of table 12 " polybase base weight is multiple " microsatellite marker multiplex PCR system
7 three base microsatellite marker specific primers are in a multiplex PCR system in the multiplex PCR system optimized
Amplification, another 5 four base microsatellite marker specific primers expand in another multiplex PCR system, and wherein primer is a pair of
The dosage (including upstream primer and downstream primer) of primer, concentration refer to final concentration, that is, the concentration after being added)
When PCR amplification, expanded using two PCR systems, i.e., 7 three base microsatellite marker specific primers are more than one
It is expanded in weight PCR system, another 5 four base microsatellite marker specific primers expand in another multiplex PCR system)
4, the paternity test of grouper stocked population:
Pick up from grouper stocked population (226 tail) and its parental population (female 15 tail of parent population, the male parent of Daya Bay, Guangdong, China
12 tail of fish), it is expanded in all individuals with the multiplex PCR system optimized, ABI3730 Genotyping instrument carries out Genotyping.
Idiotype peak figure is read using GeneMarker v 4.0, GelQuest software auxiliary carries out Data correction, interpretation and whole
Reason.
Template DNA picks up from Daya Bay, Guangdong, China, and DNA extraction method is mentioned referring to TIANGEN marine animal tissue gene group DNA
Take kit (DP324).
With Genetic relationship software PAPA v2.0 and Cervus v3.0.3 calculate site non-excluded probability (table 3) and
Accumulation excludes success rate (Fig. 3).
The non-excluded probability of 3 12, table " polybase base weight is multiple " microsatellite locus
Paternity test the result shows that: 1. only uses 5 four base microsatellite markers, has 10 stocked population individuals that can not reflect
Not, identify that accuracy rate is 95.57%；2. only uses 7 three base microsatellite markers, there are 5 stocked population individuals that can not identify,
Identify that accuracy rate is 97.79%；3. can identify parent with all 12 polybase base microsatellite markers, all filial generations, identify
Accuracy rate is up to 100% (table 4).
Table 4 is based on the grouper stocked population paternity test of " polybase base weight is multiple " microsatellite marker multiplex PCR system
5, the identification verifying of grouper stocked population:
Sanya, Hainan (38 tail), Daya Bay, Guangdong, China (41 tail) two are added in stocked population (226 tails, Daya Bay, Guangdong, China)
Wild population (wild population, which can be, releases local wild population, is also possible to the wild population in other places, wild population and
Stocked population mixes, and therefrom detects stocked population) composition separate sources population mixture, be based on paternity test technology pair
Stocked population is screened.
If the individual in stocked population is not accredited as releasing the filial generation of parent or Hainan and two, Guangdong wild population
In individual be accredited as releasing the filial generation of parent, be accordingly to be regarded as identification mistake.It is influenced to eliminate wrong bring of reading tape, by parent-offspring
The error rate of identification is set as 2e-100.
The result shows that: 1. only uses 5 four base microsatellite markers, has 44 individuals to identify mistake, identifies that accuracy rate is
85.57%；2. only uses 7 three base microsatellite markers, there are 28 individuals to identify mistake, identifies that accuracy rate is 90.82%；③.
With all 12 polybase base microsatellite markers, there are 4 individuals to identify mistake, identifies accuracy rate up to 98.68% (table 5).
The grouper stocked population that table 5 is based on " polybase base weight is multiple " microsatellite marker multiplex PCR system identifies
" polybase base weight the is multiple " microsatellite molecular marker and multiplex PCR system that the present invention obtains can accurately carry out grouper
Paternity test and group identify, therefore can be applied to the genetic breeding research of grouper and release recruitment evaluation.
The present invention is not limited within the scope of above-mentioned specific embodiment, and the embodiment above is used for the purpose of can be to this
The use process of invention is described in detail, and has the production method of equal function and technical detail to also belong in the present invention
A part of appearance.In fact, those skilled in the art are according to description above, it will be able to according to respectively needing to find different tune
Perfect square case, these adjustment all should be in the scope of the claims by the appended claims herein.
Sequence table
<110>Zhongshan University
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Claims (7)
1. a kind of grouper stocked population identifies the specific primer of microsatellite marker, it is characterized in that: the specific primer is
12 pairs, specific primer and 5 couple of four base repetitive sequence GRQU-1 including 7 couple of three base repetitive sequence GRTR-1~GRTR-7
The specific primer of~GRQU-5, wherein the base sequence of the specific primer of three base repetitive sequence GRTR-1~GRTR-7 is such as
Shown in No:1~14 SEQ ID, the base sequence such as SEQ of the specific primer of four base repetitive sequence GRQU-1~GRQU-5
Shown in No:15~24 ID.
2. a kind of discrimination method of grouper stocked population, it is characterized in that the following steps are included:
(1) grouper " polybase base weight is multiple " microsatellite marker is screened, spy described in claim 1 is designed according to microsatellite marker
Specific primer；
(2) using the specific primer in step (1), grouper " polybase base weight is multiple " microsatellite marker multiplex PCR system is established；
(3) by grouper stocked population and its parental population, using the multiplex PCR system established in step (2), in whole individuals
In expanded, then carry out paternity test；
(4) wild population composition separate sources the identification verifying of grouper stocked population: is added in grouper stocked population
Population mixture screens stocked population based on paternity test technology using the multiplex PCR system established in step (2).
3. the discrimination method of grouper stocked population according to claim 2, it is characterized in that: micro- described in step (2) defend
Asterisk remembers that multiplex PCR system includes two multiplex PCR systems, and the primer that wherein multiplex PCR system 1 uses is 7 pair of three base weight
The specific primer of complex sequences GRTR-1~GRTR-7, the primer that multiplex PCR system 2 uses is 5 pair of four base repetitive sequence
The specific primer of GRQU-1~GRQU-5.
4. the discrimination method of grouper stocked population according to claim 3, it is characterized in that: the multiplex PCR system 1
Total volume be 20 μ L, including 10 μ L, GRTR-1 specific primer of 2x Multiplex PCR MasterMix, 0.5 μ L (0.25
μM), 0.6 μ L of GRTR-2 specific primer (0.3 μM), 0.45 μ L of GRTR-3 specific primer (0.225 μM), GRTR-4 specificity
0.3 μ L of primer (0.15 μM), 0.55 μ L of GRTR-5 specific primer (0.275 μM), 0.3 μ L (0.15 μ of GRTR-6 specific primer
M), 0.2 μ L of GRTR-7 specific primer (0.1 μM), DNA profiling (100ng/ μ L) 1 μ L, ddH2O 6.1μL。
5. the discrimination method of grouper stocked population according to claim 4, it is characterized in that: the multiplex PCR system 2
Total volume be 20 μ L, including 10 μ L, GRQU-1 specific primer of 2x Multiplex PCR MasterMix, 0.3 μ L (0.15
μM), 0.35 μ L of GRQU-2 specific primer (0.175 μM), 0.25 μ L of GRQU-3 specific primer (0.125 μM), GRQU-4 are special
0.25 μ L of specific primer (0.125 μM), 0.35 μ L of GRQU-5 specific primer (0.175 μM), DNA profiling (100ng/ μ L) 1 μ L,
ddH2O 7.5μL。
6. the discrimination method of grouper stocked population according to claim 5, it is characterized in that: micro- described in step (2)
The reaction condition of satellite markers multiplex PCR system are as follows: 95 DEG C of initial denaturation 10min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C
Extend 1min, totally 35 circulations, last 72 DEG C of extensions 5min.
7. the specific primer that grouper stocked population described in claim 1 identifies microsatellite marker releases group in grouper
The paternity test of body, group identify and genetic breeding and release the application in recruitment evaluation.
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